Phage-Encoded TelN Inhibits Mre11-Rad50 to Protect Hairpin Telomeres

The ends of linear chromosomes require protection from host repair machinery that otherwise will mistake them for damaged DNA. TheE. colibacteriophage N15 harbors a linear genome with covalently closed hairpin ends formed by the phage-encoded telomere resolvase TelN. The double-strand break repair complex Mre11-Rad50 (MR) specifically targets DNA termini, posing a direct threat to N15 genome integrity, yet how hairpin telomeres evade host nuclease degradation in bacteria remains unknown. Here, we demonstrate that TelN is essential and sufficient to protect hairpin telomeres from MR processing inE. coli. Using a combination of genetic and biochemical approaches, we show that this protective function requires both TelN sequence-specific DNA binding and species-specific protein- protein interactions. Notably, we found that protection is independent of TelN’s resolution activity and does not require the C-terminal domain of TelN. Our findings reveal a potentially broad mechanism of telomere protection, providing insights into a conserved regulation of MR activity at chromosome ends across the tree of life.